Modified L-nucleic acid

ABSTRACT

The present invention relates to a modified L-nucleic acid, comprising a L-nucleic acid part and a non-L-nucleic acid part, whereby the L-nucleic acid part is conjugated to the non-L-nucleic acid part and the conjugation of the L-nucleic acid part with the non-L-nucleic acid past leads to a slowed elimination out of the organism, in comparison with a L-nucleic acid which only comprises the L-nucleic acid part, said L-nucleic acid part being a spiegelmer.

The present invention relates to modified L-nucleic acids, their use, aswell as to methods for their preparation.

Besides the use of comparatively small organic molecules the developmentof novel therapeutic concepts resorts increasingly to monoclonalantibodies, peptides and functional nucleic acids, i.e. such nucleicacids, that bind specifically to a target structure. Typical exponentsof these functional nucleic acids are the so called aptamers, that havealready been developed against a multitude of different biomolecules.Thereby, starting from a D-nucleic acid library, one or more nucleicacid molecules, the so called aptamers, that are distinguished by aparticularly high affinity towards their target structure are isolatedin several steps by in vitro selection. Methods for the preparation ofsuch aptamers are described, for example, in European patent applicationEP 0 533 838.

In pharmacology the problem of stability and biological availability,expressed as the biological half time of the administeredpharmaceutically active agents is sufficiently well known. Strategies toachieve a biological half time that permits the optimal effect of theadministered pharmaceutically active substances concentrate on one handon an appropriate modification of the pharmaceutically active agents andon the other hand on the development of appropriate forms ofadministration. In the former case considerable limitations exist, suchthat it has to be ensured that the compound having an increasedbiological half time, i.e. retention time in the organism to be treated,does not lose its pharmacological properties, in other words itsefficacy as well as causing as little as possible side effects.

Apart from the aptamers mentioned above there exists a further form offunctional nucleic acids in the so-called spiegelmers. The spiegelmerstoo bind specifically to a target sequence, wherein here though it isselected against the enantiomeric form of the target using a D-nucleicacid library, and thereupon the D-nucleic acids binding to it areprepared as L-nucleic acids, and as a result of the chiral reciprocitythese are able to bind to the true target and not to the enantiomericform thereof used for the selection process. Methods for the preparationof such spiegelmers are described, for example, in international patentapplication WO 98/08856.

Seen purely chemically, spiegelmers are L-nucleic acids, typicallyL-oligonucleotides, that virtually cannot be degraded by natural enzymesas a result of their assembly from L-nucleotides. Apart from the targetspecificity, this characteristic qualifies them for use in the mostdifferent areas, such as e.g. analysis of biological samples, diagnosisand therapy.

Similar to other chemical compounds, that have applications indiagnostics as well as therapy, particularly those which are applied inan organism, there is thus a need for spiegelmers to convert these intoa form, that permits that the spiegelmers are present and effective inan organism over an extended period of time. Thereby it is a furtherobject underlying the present invention, that the target moleculespecificity characteristic for the spiegelmers is not influenced by themodification.

According to the invention, the object is solved in a first aspect by aL-nucleic acid comprising a L-nucleic acid part and a non-L-nucleic acidpart, and the conjugation of the L-nucleic acid part with thenon-L-nucleic acid part leads to a retarded excretion from the organismand renal clearance, respectively, compared to a L-nucleic acidcomprising only the L-nucleic acid part.

In a second aspect the object underlying the invention is solved by amodified L-nucleic acid, comprising a L-nucleic acid part and anon-L-nucleic acid part, wherein the conjugation with the non-L-nucleicacid part leads to an increased retention time in an organism comparedto a L-nucleic acid comprising only the L-nucleic acid part.

In a third aspect the object underlying the invention is solved by amodified L-nucleic acid, particularly by modified L-nucleic acidaccording to the invention, comprising a L-nucleic acid part and a nonL-nucleic acid part, wherein the non-L-nucleic acid part has a molecularweight of more than about 300 Da, preferably more than about 20,000 Da,and more preferably more than about 40,000 Da.

In one embodiment of the modified L-nucleic acids according to theinvention it is intended that the L-nucleic acid part is conjugated withthe non-L-nucleic acid part, and that the non-L-nucleic acid part has amolecular weight of more than about 300 Da, preferably more than about20,000 Da, and more preferably more than about 40,000 Da.

In a further embodiment of the modified L-nucleic acids according to theinvention it is intended that the modified L-nucleic acid has amolecular weight of about 600 to 500,000 Da, preferably of about 10,000to 400,000 Da, more preferably of about 50,000 to 300,000 Da.

In yet a further embodiment of the modified L-nucleic acids according tothe invention it is intended that the L-nucleic acid part has amolecular weight of 300 to 50,000 Da, preferably of 5,000 to 25,000 Da,more preferably of 7,000 to 15,000 Da.

Finally in an embodiment of the modified L-nucleic acids according tothe invention it is intended that the non-L-nucleic acid part is linkeddirectly or indirectly to the L-nucleic acid part via a functional groupof the L-nucleic acid part, which is present on or bound to one of thefollowing components of the L-nucleic acid, wherein the functional groupis selected from the group comprising terminal and non-terminalphosphates, terminal and non-terminal sugar portions, and natural andnon-natural purine bases and natural and non-natural pyrimidine bases.

In one embodiment of the modified L-nucleic acids according to theinvention it is intended that the linkage of the non L-nucleic acid partwith the L-nucleic acid part occurs via the 2′-OH—, 3′-OH— and/or 5′-OH—group or a derivative thereof of one or more of the sugar portions ofthe L-nucleic acid part.

In a further embodiment of the modified L-nucleic acids according to theinvention it is intended that the linkage occurs via at least one of thepositions 5 or 6 of the pyrimidine base.

In yet a further embodiment of the modified L-nucleic acids according tothe invention it is intended that the linkage occurs via at least one ofthe position 8 of the purine bases.

Finally, in one embodiment of the modified L-nucleic acids according tothe invention it is intended that the linkage occurs at one or more ofthe exocyclic and/or endocyclic amine groups und/or keto groups of thepurine and/or pyrimidine bases and/or abasic position(s).

In one embodiment of the modified L-nucleic acids according to theinvention it is intended that the non-nucleic acid part is selected fromthe group comprising linear poly(ethylene)glycol, branchedpoly(ethylene)glycol, hydroxyethyl starch, peptides, proteins,polysaccharides, sterols, polyoxypropylene, polyoxyamidate,poly(2-hydroxyethyl)-L-glutamine, precise polyethylene glycol.

In one embodiment of the modified L-nucleic acids according to theinvention it is intended that a linker is arranged between the L-nucleicacid part and the non-L-nucleic acid part.

In a further embodiment of the modified L-nucleic acids according to theinvention it is intended that the L-nucleic acid part comprises anucleic acid according to SEQ ID NO. 1.

In a preferred embodiment of the modified L-nucleic acids according tothe invention it is intended that the L-nucleic acid part has an6-aminohexylphosphate at the 5′-OH end as a linker.

In particularly preferred embodiment of the modified L-nucleic acidsaccording to the invention it is intended that polyethylene glycol iscoupled to the free amine of the aminohexylphosphate linker.

In a fourth aspect the object is solved by using the L-nucleic acidsaccording to the invention as a diagnostic or diagnostic means.

In a fifth aspect the object underlying the invention is solved by theuse of the modified L-nucleic acids according to the invention for thepreparation of a medicament.

In a sixth aspect the object is solved by a method for the provision ofa modified L-nucleic acid, particularly of the nucleic acids accordingthe invention, comprising a L-nucleic acid part and a non-L-nucleic acidpart, wherein the following steps are intended:

-   -   a) providing a L-nucleic acid, which forms the L-nucleic acid        part or a part thereof of the modified L-nucleic acid;    -   b) providing a non-L-nucleic acid, which forms the non-L-nucleic        acid part or a part thereof of the modified non-L-nucleic acid;    -   c) reacting the L-nucleic acid from a) and the non-L-nucleic        acid from b); and    -   d) optionally isolating the modified L-nucleic acid obtained in        step c).

In one embodiment of the method according to the invention it isintended that the L-nucleic acid in step a) comprises a linker.

In a further embodiment of the method according to the invention it isintended that after providing the L-nucleic acid in step a), it isprovided with a linker.

It is furthermore within the scope of the present invention, that thenon-L-nucleic acid part comprises a linker and that after providing thenon-L-nucleic acid in step b), it is provided with a linker,respectively.

The present invention is based on the surprising finding, that upon theuse of L-nucleic acids in an organism as for example an mammalianorganism and in particular in a mammal that is selected preferably fromthe group comprising humans, monkeys, dogs, cats, sheep, goats, rabbits,guinea pigs, mice and rats, though L-nucleic acids are not metabolised,which goes back to the nucleases occurring in such organisms as a rule,do not recognise L-nucleic acids as a substrate due to theirstereospecificity, the biological half time of the L-nucleic acids insaid organisms is nevertheless comparatively low. So it was noticed,that upon in vivo administration of unmodified L-nucleic acids of arandom sequence in rats and monkeys the half time was between 30 minutesand 6 hours. Also, using a L-nucleic acid, i.e. a spiegelmer, that isdirected against a target molecule present in the tested organism, theabove observation of a comparatively low biological half time wasconfirmed, which confirms that this is not due to an artefact as aresult of the non-specificity of the L-nucleic acid. Furthermore thepresent inventors noticed, that the half time of non-modified L-nucleicacids is approximately as high as the half time of non-modifiedD-nucleic acids. At the same time the stability of the non-modifiedL-nucleic acids clearly increased compared to the stability ofnon-modified D-nucleic acids. Surprisingly, it could be shown, thatbecause of the modification the half time of L-nucleic acids increasesmore than upon the modification of D-nucleic acids. Thus, there is goingto be a change, completely unexpectedly, in the half time of themodified L-nucleic acids as a result of the modification compared withthe half time of the modified D-nucleic acids, whose half timesotherwise are similar to each other in the non-modified form. In otherwords, only as a result of the modification the desired effect of aprolonged half time of functional nucleic acids can be realised, whichis possible using modified L-nucleic acids only, and not using modifiedD-nucleic acids, though. In the specific case described above it is aspiegelmer for the hormone agonist gonadotropin releasing hormone(GnRH), which was administered to male, orchidectomised rats. GnRHstimulates the synthesis and release of the gonadotropins folliclestimulating hormone (FSH) and luteinising hormone (LH) There areincreased FSH- and LH levels in male, orchidectomised rats due to theabsent testosterone feedback signal. The specific spiegelmer caused aclear lowering of the LH level in a fist study (100 mg/kg, s.c.application), however after a few hours already, a reduction of theefficiency of the spiegelmer could be observed. In a performance of thesame study with the corresponding GnRH spiegelmer PEG conjugate (150mg/kg, i.v. application) a complete lowering of the LH level could beobserved, however, which still stayed completely lowered over a periodof time of 24 hours. The GnRH spiegelmer PEG conjugate represents anexample for a modified L-nucleic acid according to the invention. TheGnRH spiegelmer corresponds here to the L-nucleic acid part, and the PEGto the non-L-nucleic acid part.

These results show, that the retention time of L-nucleic acids likespiegelmers in an organism can be extended by a modification,particularly by a high-molecular modification of the L-nucleic acid. Themodification of the L-nucleic acid takes place by linking of same with anon-L-nucleic acid. Without intended to be bound by theory, thissurprising observation appears to go back to the elimination of themodified L-nucleic acid from an organism, particularly from a mammalianorganism is slowed down as a result of the increased molecular weight ofa L-nucleic acid modified in such a way. Since the elimination istypically carried out via the kidneys, it is assumed that at present theglomerular filtration rate of the kidneys regarding the modifiedL-nucleic acids is significantly reduced compared with those of thenon-modified L-nucleic acids, which leads to an increased retentiontime, i.e. biological half time of the modified L-nucleic acid comparedto the retention time of the corresponding but not modified L-nucleicacid.

Particularly remarkable in this context is the fact, that despite themodification carried out the modified L-nucleic acid, i.e. in particularthe L-nucleic acid part thereof, which is responsible for the targetmolecule specificity, obviously loses nothing of its specificity. Thus,the modified L-nucleic acids according to the invention completelysurprisingly have the characteristics, which otherwise can not normallybe realised in other pharmaceutically active compounds, namely that onecan do without extensive galenic formulations, for example in form ofdepot preparations, that release the agent successively, and rather adirect modification of the agent in question can be brought about,without its biological activity being negatively influenced, in case ofthe spiegelmers particularly expressed as specificity of the reaction orthe formation of a complex with their respective target molecule. Inother words, the modified L-nucleic acids according to the inventionovercome the incompatibility of the pharmaceutically active agent withan increase of its retention time in an organism, in particular with areduction in elimination as for example in glomerular filtration ratethat exists otherwise in pharmaceutically active agents and particularlyin small agent molecules of specific activity. Here, it is remarkablethat the affinity of the L-nucleic acid part remains essentiallyunchanged by the conjugation with the non-L-nucleic acid part.

The previously said applies of course not only in case of the use ofmodified L-nucleic acids like spiegelmers as therapeutically activeagents, but also for their use as diagnostic means, particularly totheir use as in vivo diagnostics. A typical example of the use ofspiegelmers as in vivo diagnostics is the in vivo imaging, and hereinthe use of radionucleotide carrying spiegelmers for the positronemission tomography, in particular. In this application, it is gearedtowards the radionucleotide surviving for an exactly defined period oftime. Would the radionucleotide and thus the radioactivity stay withinthe organism for a longer period of time, wherein longer is meant to betaken as necessary to carry out the respective examination, this wouldbe accompanied by an exposition to radioactive radiation unnecessary forthe patient and in some cases possibly even posing a health risk. On theother hand, in the case that die elimination of the diagnostic means andthus the radioactive label from the body occurs too fast, this wouldlead to no appropriate diagnosis or diagnostic statement being possible.With the availability of the modified L-nucleic acids according to theinvention the diagnostic means can be adjusted in an optimised mannerdepending on the respective requirements with regard to its retentiontime, i.e. its biological half time. This is largely based too on theobservation, that the glomerular filtration rate becomes severelylimited from a molecular weight of more than about 45,000 Da onwards.Otherwise the elimination, in particular the glomerular filtration rate,is clearly correlated with the size of the molecule. By using a suitablenon-L-nucleic acid part, as for example those described herein, theretention time of the modified L-nucleic acid can be adjusted exactly tothe requirements.

The chemical nature of the non-L-nucleic acid part of the modifiedL-nucleic acid can be designed virtually freely within the scope ofcertain limits. A requirement for the modified nucleic acid administeredinto an organism, which should be fulfilled in the multitude of cases ofapplication of the modified L-nucleic acid, is that the non-L-nucleicacid part consists of one or more non-immunogenic compounds.Alternatively, but if applicable also additionally thereto, these may belipophile compounds as well as hydrophile compounds. For the personskilled in the art it is obvious, that depending on the generalconditions of the isolated case also slightly immunogenic compounds maybe recruited, in particular if the administration of the modifiedL-nucleic acid according to the invention is not intended to occur overa longer period of time or repeatedly, but merely one time. In turn,this aspect is particularly of importance, if the modified L-nucleicacids according to the invention have to be administered over a longerperiod of time or repeatedly. As a rule, it will have to be ensured,that no immune response is generated by the non-L-nucleic acid part ofthe modified L-nucleic acid according to the invention upon applicationof the modified L-nucleic acid, which upon renewed administration of thesame, would lead to an immunologic or allergic reaction.

The non-L-nucleic acid part of the modified L-nucleic acid may bedesigned such that more than one non-L-nucleic acid part is bound to orconjugated with a L-nucleic acid part. For example, it is possible, thattwo or more non-L-nucleic acid parts are bound to the L-nucleic acidpart. The single non-L-nucleic acid part is preferably a polymer,wherein the subunits of the polymer may have a comparatively lowmolecular weight. It is also within the scope of the present invention,that more than one L-nucleic acid part is bound to a non-L-nucleic acidpart.

A further aspect, that has to be taken into consideration when selectingthe non-L-nucleic acid part of the modified L-nucleic acid, is theaddressing of the spiegelmers to certain compounds, particularly tocertain organs or cells. Here, depending on the specific circumstances,the non-L-nucleic acid part can be adjusted such that the modifiedL-nucleic acid accumulates preferably in certain cells, tissues ororgans, independent of the binding specificity of the spiegelmer or themodified L-nucleic acid, caused by the L-nucleic acid part.

Typically, the molecular weight of the non-L-nucleic acid part isbetween 300 and 500,000 Da. The L-nucleic acid part can be coupledeither individually, multiply, or in any combination with othernon-L-nucleic acid parts onto same or different locations of theL-nucleic acid part of the modified L-nucleic acid.

It is within the scope of the present invention, that the molecularweight of the modified L-nucleic acid is strongly determined by thenon-L-nucleic acid part. Basically, the modified L-nucleic acid may havea molecular weight from around 600 to 500,000 Da, preferably from around10,000 to 400,000 Da and more preferably from around 50,000 to 300,000Da. Lower molecular weights are realised for example by modifiedL-nucleic acids of a kind, that are cholesterol conjugates and typicallyhave a molecular weight from around 10 kDa to 25 kDa. Higher molecularweight ranges are realised for example by modified L-nucleic acids of akind, that are HES conjugates and often have a molecular weight fromaround 100 kDa to 500 kDa. In the case, that the modified L-nucleicacids are PEG conjugates, the preferred molecular weight is around 40 to70 kDa.

As a non-L-nucleic acid part can be used for example:

polyether, alkoxypolyether, as for example linear or branchedpoly(ethylene)glycols (PEG), methoxypoly(ethylene)glycols,ethoxypoly(ethylene)glycols, precise PEG (wherein precise PEG is apolyamide of the form (—NH—Y—NH—CO—X—CO—), wherein Y and Z may be variedat each location as (—CH₂CH₂O—)_(p) with different p in the range of4-6), poly(2-hydroxyethyl)-L-glutamines, polyoxypropylenes, which aredistinguished in particular by not being metabolisable in vivo andinasmuch the effect of the controlled elimination caused by the size,i.e. the molecular weight of the modified L-nucleic acid is particularlylastingly reflected and is not interfered by breakdown processes in thenon-L-nucleic acid part.

peptides, polypeptides and proteins, such as e.g. albumin, wherein thesecompounds may be naturally existing ones as well as substances addedfrom the outside.

polysaccharides, as e.g. hydroxyethyl starch, dextranes, are as far asthey are concerned metabolisable and are able to influence the retentiontime very specifically, as a result of the exactly controllabledegradation rate. The molecular weight of the hydroxyethyl starch, usedin one embodiment is between 10 kDa, preferably between 40 kDa and 400kDa, preferably between 100 kDa and 300 kDa. Hydroxyethyl starch has amolar degree of substitution from 0.1 to 0.8 and a ratio of C₂:C₆ in therange of 2 to 20. Regarding the coupling of the polysaccharides onto theL-nucleic acid part of the modified L-nucleic acid applies what was saidherein in the context of the sugar portion of the L-nucleic acidsregarding the use of the OH groups and their derivatisation.

sterols, as e.g. cholesterol. Though sterols are distinguished by arelatively low molecular weight, however, this already may lead to anincrease in the retention time of the L-nucleic acid modified in such away. Of more importance still in this respect is the behaviour of thesterols to be judged, in particular of cholesterol, which forms anon-covalent complex with lipoproteins in vivo, such as for example HDL,whereby an enlargement of the molecule and thus a longer half time isachieved.

Basically, it is within the scope of the present invention, that thenon-L-nucleic acid part is formed too from one or more D-nucleosides andD-nucleotides, respectively, wherein these may have furthermodifications, individually or as a whole, as for example modificationsfor increased stability in biological systems. Such modifications arefor example the fluoridation at the position 2′ of the sugar portion ofthe nucleotides and nucleosides, respectively. Furthermore, theseD-nucleosides and D-nucleotides may be a component of the differentnon-L-nucleic acids, particularly of those previously mentioned, butalso a part of one of the linkers described herein. Here, it is withinthe scope of the present invention, that individual or several of theD-nucleosides or D-nucleotides may comprise also one or more abasicpositions.

The linkage of the L-nucleic acid part with one or more of thenon-L-nucleic acid parts may occur on principle at all components orgroupings of the two parts assembling the modified L-nucleic acid,wherein it may be intended that derivatisations occur at one or morelocations of one or both parts, i.e. at the L-nucleic acid as well as atthe non-L-nucleic acid part(s). The linkage may occur in particular atthe 5′-OH, 3′-OH or the 2′-OH group of the L-nucleic acid, in particularat the ribose or deoxyribose part thereof.

At the same time it is also within the scope of the present invention,that at least a portion of the sugar components of the nucleotidesassembling the L-nucleic acid may have a sugar other than ribose ordeoxyribose. Such sugars may be for example further pentoses, such asfor instance arabinose, but also hexoses or tetroses or may contain alsoa nitrogen atom, as for example in a morpholino ring or aza- or thiosugar, or further sugar modifications as in locked nucleic acids (LNA)or peptide nucleic acids (PNA). These OH groups may by appropriatechemical modification be present as NH₂, SH, aldehyde, carboxylic acid,phosphate, iodine, bromine, or chlorine groups. Further functionalgroups, which allow a linkage onto the L-nucleic acid part, are known tothe person skilled in the art. As far as the linkage of non-L-nucleicacid part with a L-nucleic acid part is described herein, the commentsapply basically also for the case that more than one non-L-nucleic acidpart is linked to the L-nucleic acid part or bound to it, provided thatno statements to the contrary are given.

It is further within the scope of the present invention that at leastone part of the phosphate groups of the nucleotides assembling themodified L-nucleic acid has modification. Such modifications are forexample phosphothioates, phosphodithioates, phosphoamidates,phosphonates and further modification known to persons skilled in theart.

Apart from the linkage of the L-nucleic acid part to the non-L-nucleicacid part via the sugar portion of the L-nucleic acid part, the linkagemay occur also at the phosphate backbone, wherein here too, as pointedout in the context of the linkage via the ribose or deoxyribose part ofthe L-nucleic acid, a corresponding modification may occur. Eventually,linkages via the position 5 and/or 6 of the pyrimidine base(s), position8 of the purine base(s), as well as the exo- and endocyclical amine andketo groups of the respective nucleobases are possible, if applicablealso after functional modification of the same, as elaborated on above.Apart from natural bases the L-nucleic acid may contain one or morenon-natural bases, like e.g. isoguanidine, isocytidine, xanthosine,inosine, 2,4-diaminopyrimidine. Here it is within the scope of thepresent invention that any of the linkages described herein between theL-nucleic acid part and the non-L-nucleic acid part may occur directlyor indirectly. An indirect linkage is present in particular if a linkeris arranged between the L-nucleic acid part and the non-L-nucleic acidpart, for example a linker described herein, and provides one or both ofthe functional groups.

It is also within the scope of the present invention that between theL-nucleic acid part and the non-L-nucleic acid part(s) one or moreso-called linker may be included. Such a linker typically consists of atleast one functional group as well as a means for distance keeping or aspacer. On one hand, the function of this linker may consist infacilitating the coupling reaction. In addition or alternatively it mayimpart a function such that a spatial distance is built up between theL-nucleic acid part and the non-L-nucleic acid part of the modifiedL-nucleic acid. Such a distance is of advantage under certaincircumstances, for instance if interactions between the parts assemblingthe modified L-nucleic acid, in particular between the L-nucleic acidpart and the non-L-nucleic acid part or between two or morenon-L-nucleic acid parts of the modified L-nucleic acid are to beprevented.

In turn, the linker itself may comprise one or more functional groupsand be linked at one of the sites of the L-nucleic acid part mentionedabove to the latter. Typically the spacer consists of i.a. alkyl chainsof different length, wherein a chain length of 1 to 20, in particular of4 to 15 und further in particular 6 to 12 C-atoms is preferred. Thealkyl chain itself may be branched or carry further functional groups. Atypical embodiment of the spacer comprises ether linkages between singlemonomers, such as are present in e.g. poly(ethylen)glycol orpolyproxylene, wherein here the monomers are often present 1 to 20 timesin the polymers. Also, in forming the spacer from polyaminealkyl orpolyamidoalkyl chains a frequency of a value of 1 to 20 is common formonomers assembling these polymers.

The linker may either be coupled to one of the L-nucleotides, that formthe L-nucleic acid part of the modified L-nucleic acid. Alternatively,the linker may be included into the emerging oligomer during theenzymatic or chemical synthesis of the L-nucleic acid. Furthermore, itis within the scope of the present invention that the L-nucleic acid ismodified post synthetically und is thereby provided with a linker forthe coupling of a non-L-nucleic acid part.

It is also within the scope of the present invention that a linker isincluded for example, in an abasic position in a hairpin loop or a the3′- or 5′-end or at another position.

If the L-nucleic acid is a spiegelmer the abasic position may beincluded at a position of the spiegelmer, that is not essential forbinding of the target molecule, and for the structure of the spiegelmer,respectively. Abasic position refers here to a site of the L-nucleicacid part, that analogously to a normal nucleotide possesses the samebackbone from phosphate and sugar, but in which the nucleobase issubstituted by a hydrogen atom or a linker, as is also shown in FIG. 24.

The modified L-nucleic acids, herein referred to also as L-nucleic acidconjugates, as already evident from the itemisation above regarding thelinkage sites between the L-nucleic acid part and the non-L-nucleic acidpart may be prepared by a number of reactions, as elaborated on in moredetail in the following.

Acid amides may be prepared by reaction of N-hydroxysuccinimid (NHS) orsimilar activations of carbonic acids, such as anhydride, acid chloride,esther, succinimid and maleimide from a primary or secondaryamine^(1,2).

Thioether may be prepared starting from a halide or thiol^(3,4) and maysubsequently be subject of an oxidation into sulphoxide orsulphone^(5,6) Halides, in particular haloacetyl (iodoacetic acid,bromoacetic acid) may be coupled to any functional group of one of bothL-nucleic acid parts per ester or acidamide bond, and subsequently thehighly reactive iodine or bromine group may be coupled with a freethiol. Haloacetyl is therfore a special case of the halogenide thiolcoupling⁷. Thioether may also be prepared starting from maleimide andthiol⁷⁻⁹, isothiourea from isothiocyanate and amine¹⁰, isourea startingfrom isocyanate and amine¹¹, carbamate starting from isocyanate andalcohol¹², C—C linkages by means of a Diels-Alder reaction¹³,heterocycles by 1,3dipolar cycloaddition¹³, amines by reductiveamination, following the reaction of; for instance, aldehyde or ketonewith an amine upon subsequent reduction¹⁴, acidamide starting from anacid and an amine^(15,16), ester starting from carbonic acid or theactivated carbonic acids mentioned above and alcohol, sulphonamidestarting from amine and sulphonylchloride¹⁷, secondary amines startingfrom an epoxide and an amine^(18,19), thioether starting from an epoxideand a thiol²⁰, a disulphide starting from a thiol and a further thiol ora disulphide^(21,22), hydrazones starting from a hydrazine and analdehyde or a ketone, wherein the hydrazone may be further reduced to astable modified hydrazine²³, phosphothiates starting from a phosphate oran activated phosphoric acid, such as for example phosporoimidazolideand a thiol²⁴, phophoramidate starting from a phosphate or an activatedphosphoric acid, such as for example phosporoimidazolide orphos-N-hydroxyd benzotriazole and an amine²⁴⁻²⁷. Thereby, it is withinthe scope of the present invention to couple first a linker via aphosphoamidate or phosphothioate linkage to the L-nucleic acid part andsubsequently to the non-L-nucleic acid part. Such linkers may beethylendiamine or cysteamine, in particular.

In principle, the explanations above apply also to the case, that thereactive starting group first mentioned is arranged at the non-L-nucleicacid part as well as to case that is arranged at the L-nucleic acidpart. The corresponding modification of the L-nucleic acid in the sensethat a corresponding reactive group is provided, is known to the personsskilled in this art. The same applies to the non-L-nucleic acid part.

The term L-nucleic acid is used herein synonymously to the termL-oligonucleotide or L-polynucleotide and refers, amongst others,L-deoxyribonucleic acid as well as L-ribonucleicacid and combinationsthereof, i.e. that single or a group of nucleotides are present as RNAand the further nucleotides making up the nucleic acid are present asDNA or vice versa. Here, it is also intended that instead of deoxyriboseor ribose other sugars may form the sugar component of the nucleotide.Furthermore, the use of nucleotides with further modifications atposition 2′, is comprised, such as NH₂, OMe, OEt, OAlkyl, NHAlkyl andthe use of natural and non-natural nucleobases, as for exampleisocytidine, isoguanosine. It is thereby also within the scope of thepresent invention that the L-nucleic acid has so-called abasicpositions, i.e. nucleotides, whose nucleobase is absent. Such abasicpositions may be arranged within the nucleotide sequence of theL-nucleic acid as well as at one or both of the ends, i.e. the 5′-and/or the 3′-end.

It is further within the scope of the present invention that theL-nucleic acid contains one or more D-nucleosides or D-nucleotides.Here, the one or the several D-nucleosides or nucleotides may bearranged within the L-nucleic acid as well as at one or both of the endsof the L-nucleic acid. The single D-nucleoside or D-nucleotide may carryone or more modifications, for example for increasing the stability ofthe nucleoside or the nucleotide, respectively, and its binding to theL-nucleic acid, respectively.

In principle, the L-nucleic acid may be present double or singlestrandedly. Typically, it is a single stranded L-nucleic acid, whichmay, however, form defined secondary structures and thus tertiarystructures also, due to its primary sequence. In the secondary structurea multitude of L-nucleic acids has double stranded sections.

Apart from the high molecular modifications described herein inparticular, L-nucleic acids may also refer to modifications with regardto single nucleotides of the nucleic acid, wherein here e.g. the 2′-OHgroup of the sugar portion of the nucleotides may be present as amethylether, as already disclosed above.

The L-nucleic acids and L-nucleic acid parts, respectively, describedherein are preferably functional nucleic acids. To the functionalnucleic acids belong, amongst others, aptamers, spiegelmers, ribozymesand aptazymes. Preferably, the L-nucleic acids and L-nucleic acid parts,respectively, are spiegelmers. As mentioned already in the beginning,spiegelmers are nucleic acids that bind to a target molecule or a partthereof and are made up from L-nucleotides, at least in the part of thenucleic acid binding to the target molecule. Preferably, they are theresult of contacting a nucleic acid library, in particular a statisticalnucleic acid library, with the target molecule.

For a selection method for the development of functional nucleic acidscombinatorial DNA libraries are prepared first. As a rule, it is asynthesis of DNA oligonucleotides, that contain a region from 10-100randomised nucleotides in the center, which is flanked 5′-und3′-terminally by two primer binding sites. The preparation of suchcombinatorial libraries is described, for example, in Conrad, R. C.,Giver, L., Tian, Y. and Ellington, A. D., 1996, Methods Enzymol., Vol267, 336-367. Such a chemically synthesised single stranded DNA librarycan be converted into a double stranded library by the polymerase chainreaction, which may be used for a selection by itself. As a rule, aseparation of the individual strands takes place with suitable methods,such that a single stranded library is regained, which is used for theselection method, if it is a DNA selection (Bock, L. C., Griffin, L. C.,Latham, J. A., Vermaas, E. H. und Toole, J. J., 1992, Nature, Vol. 355,564-566). It is still just as possible to use the chemically synthesisedDNA library directly for the in vitro selection. In addition, an RNAlibrary may, in principle, be generated from double stranded DNA, if aT7 promoter has been included previously, also by a suitable DNAdependant polymerase, e.g. T7 RNA polymerase. Aided by the methodsdescribed, it is possible to generate libraries of 10¹⁵ and more DNA orRNA molecules. Every molecule from this library has a different sequenceand thus a different three-dimensional structure. By the in vitroselection methods it is now possible to isolate one or more DNAmolecules from the mentioned library, that have a significant bindingproperty towards a given target, by one or more cycles of selection andamplification as well as mutation, if applicable. The targets may beviruses, proteins, peptides, nucleic acids, small molecules such asmetabolites of the metabolism, pharmaceutical agents and metabolitesthereof, or other chemical, biochemical or biological compounds, such asdescribed, for example, in Gold, L., Polisky, B., Uhlenbeck, O. undYarus, 1995, Annu. Rev. Biochem. Vol. 6, 763-797 and Lorsch, J. R. andSzostak, J. W., 1996, Combinatorial Libraries, Synthesis, Screening andapplication potential, ed. Riccardo Cortese, Walter de Gruyter, Berlin.The prodcedure is performed in such a manner, that binding DNA or RNAmolecules are isolated from the library initially used, and areamplified after the selection step by means of polymerase chainreaction. In RNA selection a reverse transcription is to be placed aheadof the amplification step by polymerase chain reaction. A libraryenriched after a first round of selection may be used for a renewedround of selection, such that the molecules enriched in the first roundof selection have a chance to prevail again by selection andamplification and go into a further round of selection with even moredaughter molecules. At the same time the step of the polymerase chainreaction presents the possibility to introduce new mutations duringamplification, e.g. by varying the salt concentration. After sufficientrounds of amplification and selection the binding molecules prevailed.An enriched pool emerged this way, whose members may be separated bycloning, and subsequently determined with regard to their primarystructure by common methods for determining a sequence. The obtainedsequences are then tested for their binding properties with regard tothe target. The method for the generation of such aptamers is alsoreferred to as SELEX method and described, for example, in EP 0 533 838,the disclosure of which is hereby included by reference.

The best binding molecules may be shortened to their essential bindingdomain by shortening of the primary sequences, and prepared by chemicalor enzymatical synthesis.

A special form of aptamers manufacturable in such a manner are theso-called spiegelmers, which are characterised essentially by beingassembled at least partially, preferably completely, from thenon-natural L-nucleotides. Methods for the preparation of suchspiegelmers are described in PCT/EP 97/04726, whose disclosure isincluded hereby by reference. The specific feature of the methoddescribed therein, is the generation of enantiomeric nucleic acidmolecules, i.e. of L-nucleic acid molecules, that binds to a nativetarget, i.e. being in its natural form or configuration, or such atarget structure. The in vitro selection method described above is usedto select binding nucleic acids or sequences initially against theenantiomers, i.e. the non-natural structure of a natural target, as forinstance in case that the target molecule is a protein, against aD-protein. The resulting binding molecules obtained this way (D-DNA,D-RNA, and corresponding D-derivatives, respectively) are determined asto their sequences and the identical sequence is then synthesised withmirror-image nucleic acid modules (L-nucleotides and L-nucleotidederivatives, respectively). The resulting mirror-imaged enantiomericnucleic acids (L-DNA, L-RNA, and corresponding L-derivatives,respectively), so-called spiegelmers, have for reasons of symmetry amirror-imaged tertiary structure and thus a binding property for thetarget present in its natural form or configuration.

The target molecules described above, also referred to as target, may bemolecules or structures, such as e.g. viruses, viroids, bacteria, cellsurfaces, cell organelles, proteins, peptides, nucleic acids, smallmolecules such as metabolites of the metabolism, pharmaceutical agentsand metabolites thereof, or other chemical, biochemical or biologicalcompounds.

In the following, the invention is illustrated further by the figuresand examples from which further advantages, embodiments and features ofthe invention ensue.

FIG. 1 shows an hexylamine linker, that has a linear spacer (“means fordistance keeping”) consisting of six carbon atoms as well as a terminalamino group and a terminal phosphate residue. The substitution denotedwith R may present here also a nucleic acid, and the L-nucleic acid partof a modified L-nucleic acid, respectively. Via the amino group thenon-L-nucleic acid part may be coupled onto the L-nucleic acid part andthus the modified L-nucleic acid according to the invention be formed.

FIG. 2 shows further linkers, wherein the structures referred to as (2),(4), and (6) correspond to linker according to (1), (3) and (5), whereinin the latter the phosphate part provided with residue R representspreferably the L-nucleic acid part, and the non-L-nucleic acid part ofthe modified L-nucleic acid is coupled via the functional group called Xonto the L-nucleic acid. The term “oligo” stands exemplaryly for anoligonucleotide, wherein it is within the scope of the presentinvention, that this and the L-nucleic acid or the L-nucleic acid parts,respectively, herein may be generally L-polynucleotides. The varioussubstituents refer herein to the following reactive groups, that areindividual and each independent from each other:

X=OH, NH₂, HS, Hal, CHO, COOH

Y=O, NH, NMe, S, CH₂

Z₁, Z₂, Z₃, Z₄, Z₅ and Z₆=H, Me, Alkyl, HO(CH₂)_(n), HO, H₂N(CH₂)_(n),H₂N, F, wherein n is an integer between 1 and 20 and wherein Alkylrefers to linear and branched hydrocarbon chains with preferably 1-20C-atoms, more preferably 1 to 4 C-atoms and/or —(CH₂)_(n)H,—CH[(CH₂)_(n)H][(CH₂)_(m)H], —C[(CH₂)_(n)H][(CH₂)_(m)H][(CH₂)₁H],—(CH₂)_(n)(CH)_(m)[(CH₂)₁H][(CH₂)_(k)H],—(CH₂)_(n)(C)_(m)[(CH₂)₁H][(CH₂)_(k)H][(CH₂)_(j)H], wherein n, m, 1, kund j are integers independent from each other between 1 and 8,preferably 1 to 4 C-atoms.

FIG. 3 shows an overview of different linkers, that are coupled todifferent positions of the nucleobases. Thereby, it is notable that thesugar portion of the nucleoside shown in each case may be a ribose, adeoxyribose or a modified ribose and modified deoxyribose, respectively,and the residue X may be=H, HO, H₂N, MeO, EtOH or alkoxy. Thereby,alkoxy, in particular, refers to linear and branched oxyhydrocarbonchains with 1-20 C-atoms, preferably 1 to 4 C-atoms and/or —O(CH₂)_(n)H,—CH[(CH₂)_(n)H][(CH₂)_(m)H], —OC[(CH₂)_(n)H][(CH₂)_(m)H][(CH₂)₁H],—O(CH₂)_(n)(CH)_(m)[(CH₂)₁H][(CH₂)_(k)H],—(CH₂)_(n)(C)_(m)[(CH₂)₁H][(CH₂)_(k)H][(CH₂)_(j)H], wherein n, m, 1, kund j are integers independent from each other between 1 and 8,preferably 1 to 4 C-atoms. The actual linker structure is X₁-[X]_(n) inall four nucleosides (1), (2), (3) and (4) shown, wherein n is aninteger between 0 and 20. X₁ represents a functional group that isselected from the group comprising HO, H₂N, HRN, HS, SSR, Hal, CHO,COOH, COOR and COHal. In the linkers denoted as structural formulas(5-12) n is an integer between 0 and 20 as well, and Z meansindependently from other sustituents either O, NH, NR or S, wherein Rstands for alkyl as defined herein.

FIG. 4 shows possible linkers at position 5 of pyrimidine nucleosidesand nucleotides, respectively. With regards to residues R′, R″ and R′″basically applies what was said herein in the context of FIG. 5. Thelinker R has the structure —[Y]_(n)—X₁ and may preferably acquire theforms shown in structures (2) to (9), wherein Z may mean O, NH, NHR or Shere too, independently of the choice of the other substituents, and nmay be an integer between 1 and 20. The functional group X₁ ispreferably selected from the group that has HO, H₂N, HRN, HS, SSR, Hal,CHO, COOH, COOR, COHal.

FIG. 5 shows in 1 the basic structure of cytosine, which may havedifferent linker structures at its exocyclic amine. Thereby R′ refers toa L-nucleic acid or a L-polynucleotide, OH, or phosphate, R″ to aL-nucleic acid or a L-polynucleotide, OH or phosphate and R′″ to H, OH,OMe, OEt, NH₂. The residue R refers thereby to the linker, which has thebasic structure —[Y]_(n)—X₁ and may have the structural formulas shownin (2) to (9), wherein Z=O, NH, NR, S and n may be an integer between 1and 20. The functional group X₁ is preferably selected from the groupthat has HO, H₂N, HRN, HS, SSR, Hal, CHO, COOH, COOR, COHal.

FIG. 6 shows the formation of a modified L-nucleic acid according to theinvention by reaction of PEG-NHS with a L-nucleic acid provided with alinker. After successful coupling the modified L-nucleic acid ispresent, that comprises in the present case PEG as the non-L-nucleicacid part and in this actual case an oligonucleotide as L-nucleic acid,wherein a linker or a spacer, respectively, carrying an amino group isinserted between both, and it comes to an acid amid binding between thelinker and the PEG. Apart from the modified L-nucleic acid theN-hydroxysuccinimide split off the PEG is obtained as a further reactionproduct. As possible residues R, H, CH₃ and in general alkyl chains witha length of 1-20 are preferred. The functional group may in principle bethe product of any one of the reactions explained herein above. Insofar,the embodiments of the linker described in association with the furtherfigures, in particular FIGS. 2 and 3, apply also to this context. Thesame applies to the substituents and control variables like n depictedin the formula.

FIG. 7 shows the conversion of different PEG derivatives with differentlinkers. Here, the two reactions (1) and (2) differ only in that inreaction (1) the carboxyl group is present at the PEG and in reaction(2) the carboxyl group is present at a L-oligonucleotide provided with alinker. The functional group of the respective corresponding reactionpartner, i.e. in the case of reaction (1) the L-nucleic acid providedwith a linker, and in the case of reaction (2) the PEG provided with anamine group. Thus the statement is confirmed, that was made in thecontext of the different reactions as above, which are possible betweena L-nucleic acid part and a non-L-nucleic acid part, if applicable withparticipation of one or more inserted linkers, that in principle thementioned reactive groups may be present in all reaction partners thatare involved. The finally obtained structures will differ from eachother correspondingly, so that in case of reaction (1), where an acidamide group is present at the PEG, and in the case of reaction (2),where the acid amide binding is present at the construct from linker andoligonuleotide, i.e. the L-nucleic acid. With regard to the substituentsR applies what was said in the context of FIG. 6, correspondingly.

FIG. 8 shows the reaction of a halogenide with a thioester, that areattached either to the non-L-nucleic acid part or to the L-nucleic acidpart, respectively. In the reactions (1) to (3) it is intended that theL-nucleic acid, here as in all figures abbreviated as oligo, is providedwith a linker, and that the linker carries a halogenide, as for exampleI, Br, Cl. This derivatised L-nucleic acid is thereupon reacted with aPEG provided with a thiol group, preferably a terminal thiol group. Inthe case of reaction (1) a thioether bond between linker and PEG willoccur. By oxidation it may result, as depicted in reaction (2), in theformation of a sulphoxide or a sulphone, respectively. In the reactionsaccording to (4) to (6) also a reaction between a thiol and a halogenideoccurs, wherein in these cases the L-nucleic acid is provided with thethiol group, and the linker carries the halogenide. Correspondingly, aformation of compounds occurs, wherein the sulphur is arranged betweenthe L-nucleic acid and the linker, and it may be oxidised, as shown inthe reactions (5) and (6), again into the corresponding derivatives.

FIG. 9 shows the reaction of the PEG provided with a maleimide groupwith a L-nucleic acid, there referred to as oligo, that has an linkercarrying a thiol group. The reaction product is a thioether.

FIG. 10 shows the reaction of a L-nucleic acid carrying a phosphategroup with a PEG, which is provided with a linker carrying a thiolgroup. The reaction product is a phosphothioate.

FIG. 11 shows the reaction of a L-nucleic acid provided with a phosphateresidue, terminal if applicable, with a PEG, which is provided with alinker having an amine. The reaction product is a phosphoamidate.Regarding the residue R it applies what was elaborated on in the contextof FIG. 6.

FIG. 12 shows the insertion of a reactive amino or thiol group,respectively, into a L-nucleic acid using an activate phosphate group,preferably a terminal phosphate group of the L-nucleic acid. Here, aphosphorimidazolide (I) is made in a first step, which leads to theformation of a 2-aminoethylene-1-phosphoramidate (II) in the case ofreaction (2) using an ethylenediamine, or in the case of the reaction(3) using cysteamine to 2-thioethylene-1-phosphoamidate (III),respectively. The compounds according to (II) and (III) may be reactedthereupon with non-L-nucleic acids, particularly with those disclosedherein.

FIG. 13 shows the reaction of a PEG provided with a sulphonyl chloridegroup with a L-nucleic acid that has a linker carrying an amine group.The reaction product is a sulphonamide. Regarding the residue R itapplies what was elaborated on in the context of FIG. 6.

FIG. 14 shows the reaction of a PEG provided with an epoxide group witha L-nucleic acid that has a linker carrying an amine group forming anamine. Regarding the residue R it applies what was elaborated on in thecontext of FIG. 6.

FIG. 15 shows the reaction of a PEG provided with an epoxide group witha L-nucleic acid that has a linker provided with a thiol group. Thereaction product is a thioether.

FIG. 16 shows the reaction of a PEG provided with an isothiocyanategroup with a L-nucleic acid that has a linker carrying an amine group.The reaction product is an isothiourea. Regarding the residue R itapplies what was elaborated on in the context of FIG. 6.

FIG. 17 shows the reaction of a PEG provided with an isocyanate groupwith a L-nucleic acid that has a linker carrying an amine group formingan isourea. Regarding the residue R it applies what was elaborated on inthe context of FIG. 6.

FIG. 18 shows the reaction of a PEG provided with an isocyanate groupwith a L-nucleic acid that carries a free OH group, that may directlycome from the L-nucleic acid, as for example from a phosphate group orthe sugar moiety of the nucleoside, i.e. the positions 2′-OH, 3′-OH, or5′-OH. Alternatively, the OH group may be linked to the L-nucleic acidvia a suitable linker. The reaction product is a carbamate.

FIG. 19 shows the reaction of an aldehyde or keto group with an aminogroup, which is present in each case either at the non-L-nucleic acidpart (reaction (1)), in the case shown at PEG; or at the L-nucleic acidpart (reaction (2)). Preferably here, the L-nucleic acid part has alinker carrying the respective reactive group, i.e. the amino group orthe carbonyl group. In the case of the reaction (1) the PEG carries aamino group, whereas the L-nucleic acid has a linker carrying thecarbonyl group. The reaction product obtained directly, imine, isconverted thereupon into an amine by reduction. In case of the reaction(2) the PEG carrying a carbonyl group is reacted with L-nucleic acid,that carries a linker having an amino group. The reaction product imineis reduced and leads to an amine. Regarding the residue R it applieswhat was elaborated on in the context of FIG. 6.

FIG. 20 shows the reaction of a PEG provided with a thiol group with aL-nucleic acid carrying a linker provided with a thiol group as well.The reaction product is a modified L-nucleic acid, that has a disulphidegroup between the PEG and the L-nucleic acid, strictly speaking thelinker attached to it.

FIG. 21 shows the reaction of a PEG provided with a hydrazine group witha L-nucleic acid that carries a linker comprising a carbonyl group. In afist step of the reaction a hydrazone is obtained, which is thereuponconverted reductively into a substituted hydrazine. Regarding theresidue R it applies what was elaborated on in the context of FIG. 6.

FIG. 22 shows in reaction (1) the conversion of a PEG provided with aconjugated diene with a L-nucleic acid that carries a linker with aso-called dienophilic group. The dienophile consists of a C—C-doublebond, which in turn has a substituent Z comprising anelectron-withdrawing group. These may be preferably NO₂, CH₂Cl, COOR, CNor maleimide. Regarding the residue R it applies what was elaborated onin the context of FIG. 6. Due to this reaction the formation of amodified L-nucleic acid occurs that has a hexeneyl group between the PEGand the L-nucleic acid provided with a linker. The Diels-Alder reactionshown in reaction (2) starts with a PEG which has a dienophile with-asubstituent Z that reacts with a L-nucleic acid comprising a linkerwhich carries a conjugated diene. Regarding the substituent Z it applieswhat was elaborated on, in the context of reaction (1). The reactionproduct in this reaction (2) is also a L-nucleic acid conjugate linkedvia a hexeneyl group.

FIG. 23 shows the structure of the branched and linear mPEG-NHS esterthat were used.

FIG. 24 shows in (1) the basic assembly of an abasic L-nucleoside, whichinstead of the nucleobase may have either a hydrogen atom or one or moreoptionally different linker structures. Here R′ denotes a L-nucleic acidor a L-polynucleotide, OH or phosphate, R″ a L-nucleic acid or aL-polynucleotide, OH or phosphate and X=H, OH, OMe, OEt, NH₂. Theresidue R denotes either the hydrogen atom instead of the nucleobase orthe linker, which may have the structural formulas shown in (2) to (8),wherein Z=CH₂, O, NH, NR, S and n may be an integer between 1 and 20.The functional group X₁ is preferably selected from the group that hasHO, H₂N, HRN, HS, SSR, Hal, CHO, COOH, COOR, COHal.

FIG. 25 shows an activity test of a PEGylated DNA spiegelmer bindingGnRH in male orchidectomised rats.

FIG. 26 a shows an activity test of a PEGylated DNA spiegelmer bindingGnRH in vitro.

FIG. 26 b shows an activity test of a non-PEGylated and PEGylated DNAspiegelmer binding GnRH in vitro.

FIG. 27 shows the pharmacokinetics of a PEGylated DNA spiegelmer bindingGnRH in rats.

FIG. 28 a shows a pharmacokinetical profile of PEGylated L-RNA afterintravenous dose in rats.

FIG. 28 b shows a pharmacokinetical profile of non-PEGylated L-RNA afterintravenous dose in rats.

FIG. 28 c shows a pharmacokinetical profile of PEGylated L-RNA aftersubcutaneous dose in rats.

FIG. 28 d shows a pharmacokinetical profile of non-PEGylated L-RNA aftersubcutaneous dose in rats.

FIG. 29 shows an activity test of a DNA spiegelmer binding GnRH in maleorchidectomised rats in vivo.

EXAMPLE 1 Synthesis of PEG Conjugates of L-nucleic Acids

The conditions for the synthesis of PEG conjugates of L-nucleic acidswere examined starting from the L-nucleic acid depicted in SEQ ID NO: 2and PEG, wherein the PEG was modified such that it was present either asa NHS ester or as a primary amine for the coupling onto an amine and aphosphate, respectively. It was proceeded in a way that the nucleic acidwas dissolved in an aqueous system. The pH was adjusted to pH 6.5-9.0 bydifferent buffers or bases like, for example NaHCO₃, NaH₂PO₄/Na₂HPO₄,HEPES, MOPS, NH₄OAc, triethylamine. The influence of addition ofdifferent organic solvents, as for example DMF, DMSO, acetonitrile andothers was tested, wherein the portion of the organic solvent was variedbetween 0-100%. Subsequently the addition of different PEG derivativesoccurred, as for example branched mPEG₂-NHS ester, linear mPEG-NHS esteror mPEG-NH₂ (Shearwater Corporations) of different molecular weightsbetween 10,000 Da und 40,000 Da. The addition of PEG-NHS ester may bedone in different ways. Thus, PEG-NHS ester may be dissolved for examplein an acid of low concentration such as, for example 0.01 N HC1, or maybe added in drops being dissolved in an organic solvent such as DMF oradded as a solid. The preferred way of adding PEG-NHS is as a solid inportions. Further, the influence of the reaction temperature between 4°C.-65° C. was tested. As nucleic acids were used nucleic acids with thefollowing sequence 5′-NH₂-TAT TAG AGA C-3′ (SEQ ID NO: 2), and5′-PO₄-TAT TAG AGA C-3′ (SEQ ID NO: 3) as well as the nucleic acidaccording to SEQ ID NO: 1. The yields of the reactions summarised abovewere between 5-78%.

The preferred variant of reaction was the addition of two equivalentseach of solid PEG-NHS ester in intervals of around 30 minutes, six timesalltogether, to a nucleic acid dissolved in a solvent consisting of 60parts H₂O and 40 parts DMF adding NaHCO₃ (0.2 M), a pH of 8.0 and 37°.The reaction conditions lead to a yield of 78%.

EXAMPLE 2 Synthesis of a PEG Conjugate of a L-Nucleic AcidPhosphoamidate

Starting from a L-nucleic acid with the sequence 5′-P0₄-TAT TAG AGA C-3′(SEQ ID NO: 3) a corresponding phosphoamidate PEG conjugate was made.The L-nucleic acid (10 OD) was reacted with PEG-NH₂ (20,000 Da, linear,1-10 equivalents) in aqueous solution with EDCI at 50° C. to a PEGconjugate of a L-nucleic acid phosphoamidate. The analysis andpurification was done analogously to that of the PEGylation of L-nucleicacids with PEG-NHS, as described in example 1. The reaction conditionswere not optimised and led to a yield of <8%.

EXAMPLE 3 PEGylation of a GnRH Spiegelmer Ligand

The peptide hormone GnRH I (gonadotropin releasing hormone,gonadoliberine), which is generally referred to as GnRH, is adekapeptide made in the hypothalamus which stimulates the secretion ofthe gonadotropin hormones luteinising hormone and follicle stimulatinghormone (FSH) by the pituitary gland. GnRH is secreted from the neuronsof the hypothalamus in a pulsating manner and then binds to a receptoron the cell surface of the pituitary gland. The ligand receptor complexis internalised, whereby a release of FSH and LH occurs, which in turnstimulate the production of sexual hormones such as estrogen,progesteron or testosteron. A spiegelmer, i.e. a L-nucleic acid could beproduced that binds specifically to GnRH and has the following sequence:

(SEQ ID NO: 1) 5′-CCA AGC TTG CGT AAG CAG TCT CCT CTC AGG GGA GGT TGGGCG GTG CGT AAG CAC CGG TTT GCA GGG G-3′

The synthesis of the spiegelmer of the sequence shown above wasperformed on an Amersham Pharmacia Biotech Oligopilot II DNA synthesiserin 780 μMol scale on a 1,000 Å CPG solid phase (Controlled Pored Glass)according to the 2- cyanoethyl-phosphoramidit chemistry (Sinha et al.NAR, 12, 1984, p. 4539ff). Subsequently, a6-(monomethoxytritylamino)-hexyl-(2-cyanoethyl)-(N,N-diiospropyl)-phosphoramiditwas linked to the 5′ end of the spiegelmer (5′-MMT-aminohexylspiegelmer), to allow the post-synthesis conjugation with PEG.

After completion of the synthesis the 5′-MMT aminohexyl spiegelmer wascleaved from the solid phase by an 8 hour incubation in 33% ammoniasolution at 65° C., and deprotected completely, afterwards concentratedto dryness, taken up into 10 mM NaOH and purified by means of RP-HPLC.The cleavage of the monomethoxytrityl protection group occurred with0.4% trifluoracetic acid (TFA) in 30 min at RT. TFA was removed bytwofold coevaporation with ethanol and the 5′-aminohexyl spiegelmeraccording to SEQ ID NO: 1 was purified by precipitation in ethanol(yield: 5,000 OD, 7.5 μmol). The product peak was collected and desaltedby means of size-exclusion chromatography via a Sephadex G10 column orby ultrafiltration (Labscale TFF System, Millipore).

The GnRH spiegelmer 5-amino-modified in such a way (5,000 OD, 7.5 μmol)was prepared in 0.2 M NaHCO₃, pH 8.5/DMF 60:40 (v/v) (125 mL), warmed to37° C. and powdery N-hydroxysuccinimidyl (NHS) activated ester ofbranched 40.000 Da poly(ethylen)glycol was added in portions (2 eq(equivalents) every 30 min, alltogether 12 eq, (6×600 mg, 180 μmol). Theprogress of the reaction was monitored by analytical gelectrophoresis(8% polyacrylamide, 8.3 M urea). The raw product was purified initiallyby ion exchange HPLC from excess PEG (Source Q 30; solvent A: H₂O,solvent B: 2 M NaCl; low rate 20 mL/min; loading of the column andelution of free PEG with 10% B; elution of the PEG-GnRH spiegelmerconjugate with 50% B), subsequently GnRH spiegelmer PEGylated by RP-HPLCwas separated from non PEGylated GnRH spiegelmer (Source RPC 15; solventA: 100 mM triethylammonium acetate (TEAA), solvent B: 100 mM TEAA inH₂O/acetonitril 5:95; flow rate 40 mL/min; loading of the column with10% B; gradient from 10% to 70% B in 10 column volumes, elution ofPEG-GnRH spiegelmer at 45-50% B), salt exchanged (Source Q 30; solventA: H₂O, solvent B: 2 M NaCl; flow rate 20 mL/min; loading of the columnand elution of free PEG with 10% B; elution of PEG-GnRH spiegelmer with50% B) and subsequently desalted by gel filtration (Sephadex G10;solvent H₂O; flow rate 5 mL/min) or ultrafiltration (Labscale TFFSystem, Millipore). By lyophilisation the desired product was obtainedas a white powder (3.900 OD, 375 mg, 78%).

Analogously, further nucleic acids including the sequence according toSEQ ID NO:1 linked with different PEG (linear 10,000 Dalton, linear20,000 Dalton, branched 20,000 Dalton, linear 35,000 Dalton), andpurified.

EXAMPLE 4 Synthesis of FITC Conjugates of L-Nucleic Acids: Coupling ofFluorescein Isothiocyanate onto a GnHR Spiegelmer with a 5′NH₂—C₆ Linker

The 5′amino-modified GnRH spiegelmer made according to example 3 wasprepared in 0.5 M NaHCO₃ pH 8.5, warmed to 65+ C. and an excess offluorescein isothiocyanate (FITC, 10 eq) was added to the reactionmixture. The reaction was monitored by means of analytical RP-HPLC. Itwas shaken for 48 h at 65° C., excess FITC separated by Centri-Spin10(Princeton Separations) and the fluorescein labeled L-nucleic acid waspurified with RP-HPLC. Lyophilisation delivered the desired product as ayellowish powder in quantitative yield.

EXAMPLE 5 Activity Test of a GnRH Binding, PEGylated DNA Spiegelmer invivo in Male Orchidectomised Rats

Male rats were orchidectomised, whereby the LH level of the ratsincreased steadily during the following eight days due to the missingtestosteron feedback signal. On day 8 the PEG-GnRH DNA spiegelmer, i.e.the conjugate from PEG and GnRH spiegelmer, was administeredintravenously to seven rats (150 mg/kg). Blood samples were taken on day0 (prior to the orchidectomy), on day 8 (0 hours prior to i.v.application of the PEG-GnRH spiegelmer), 0.5 h, 1.5 h, 3 h, 6 h as wellas 24 h post i.v. application and the respective LH level determinedusing radioimmunoassay (RIA). In parallel, only the vehicle (PBS buffer,pH 7.4) i.v. as a negative control was administered to seven maleorchidectomised rats, and the standard antagonist Cetrorelix (100 μg/kg)s.c. as a positive control to seven male orchidectomised rats. Theresult is shown in FIG. 25.

With the exception of the negative control (in FIG. 25 depicted astriangles) there is a LH level even after 24 h under the influence ofthe PEG-GnRH spiegelmers, that is comparable to that of thenon-orchidectomised rats, and those rats, respectively, which hadreceived the standard antagonist Cetrorelix. This proves the suitabilityof the PEG-GnRH DNA spiegelmer, to influence lastingly the effect of theGnRH over an extended period of time. That the effect of the PEG-GnRHDNA spiegelmer described above is due to the PEGylation of the GnRHspiegelmer results from the fact that upon application of the GnRHspiegelmer without the corresponding modification with subcutaneousapplication of 100 mg/kg a reduction of the activity of the GnRHspeigelmer could be observed already after a few hours. The result isshown in FIG. 29 as well.

EXAMPLE 6 Activity Test of GnRH Binding, PEGylated and Non-PEGylated DNASpiegelmers in CHO Cells in vitro

The cell culture study described herein was performed on Chinese HamsterOvary (CHO) cells, which express the human receptor for GnRH. Here theintracellular release of Ca²⁺ ions was measured, since this release,important for the signal transduction, occurs after formation of theagonist receptor complex. The Ca²⁺ level was then deterined by a Ca²⁺sensitive fluorescence dye. The PEG-GnRH DNA spiegelmer and the GnRHspiegelmer, respectively, was to capture the agonist GnRH and thusinhibit its binding to the receptor on the cell membrane. It was doneexperimentally such that the agonist GnRH (2 nM) was preincubated for 20min with the GnRH spiegelmer and the PEG-GnRH DNA spiegelmer,respectively, in a concentration range of 100 pM bis 1 μM. This solutioneach was given to the CHO cells loaded with fluorescence dye, and therespective Ca²⁺ concentration determined with a Fluroescence ImagingPlate Reader (FLIPR). The result of the PEG-GnRH DNA spiegelmer (filledtriangles) and of a standard antagonist (filled squares), used here as apositive control, is shown in FIG. 26 a.

The concentration dependant determination resulted in a sigmoidalactivity curve, which indicates that the native, i.e. the non-modifiedGnRh spiegelmer (filled squares), as well as GnRH-DNA spiegelmermodified with PEG (filled triangles) were able to inhibit the formationof the GnRH receptor complex at 100%. The IC₅₀ was 20 nM for the GnRHspiegelmer und 30 nM for the PEG-GnRH DNA spiegelmer (FIG. 26 b).

EXAMPLE 7 Pharmacokinetics of a GnRH Binding PEGylated DNA Spiegelmer inRats

Seven male Wistar rats (Tierzucht Schönwalde GmbH, Germany, weight:250-300 g) were used for the determination of the phamacokineticalcharacteristics of the GnRH binding PEGylated DNA spiegelmer. The groupwas treated in parallel with the groups for the activity tests (seeexample 6), i.e. castrated after an adaption phase, and after anotherweek the animals received a single dose of 800 nmol/kg PEG-GnRH DNAspiegelmer administered intravenously. The substance was dissolved in1×PBS, pH 7.4 (stock solution: 1 mM).

For analysis blood samples were taken prior to substance dose (0 h) aswell as 1 h, 6 h, and 8 h post substance dose, and analysed as EDTAplasma.

From the plasma GnRH binding PEGylated DNA spiegelmer was extracted bysolid-phase extraction aided by weak anion exchangers. For this 50 μlEDTA plasma each were dissolved in buffer A (50 mM NaH₂PO₄, pH 5.5; 0.2M NaClO₄; 20% (v/v) formamide und 5% (v/v) acetonitril) in a totalvolume of 1 ml and stored at 4° C. over night or at −20° C. for 4 daysmaximum, respectively, until extraction. Frozen samples were thawed forat least 2 h at room temperature, mixed and subsequently centrifuged.

For solid-phase extraction dimethylaminopropyl-anion exchanger columns(DMA 3 ml/200 mg column material, Macherey & Nagel, Düren) on a Bakerspe-12G vakuum apparatus (Mallinckrodt Baker, Griesheim) was used. Thebuffers used consisted of: buffer A (50 mM NaH₂PO₄, pH 5.5; 0.2 MNaClO₄; 20% (v/v) formamide und 5% (v/v) acetonitril and buffer B (80 mMNaH₂PO₄, pH 6.0; 50 mM Na₂HPO₄, 2 M NaClO₄; 20% (v/v) formamide und 5%(v/v) acetonitril), wherein the two buffers A and B were mixed in aspecific ratio for the preparation of the wash and the elution buffer,such that the desired salt concentrations were achieved. The anionexchangers were flushed with 2 ml of buffer A. The samples were addedapplying −100 mbar and washed with 2 ml of buffer A as well as 2 ml ofwash buffer (0.4 M NaClO₄). After drying the column material for 5 minby applying −200 mbar, the PEGylated GnRH binding DNA spiegelmer waseluted with 3×0.5 ml elution buffer (0.9 M NaClO₄), wherein the bufferwas heated to 70° C. prior to elution. The eluates were stored at 4° C.until gel filtration.

As an internal standard an 30 mer DNA spiegelmer had been added to thesamples prior to extraction, which was bound to a 40 kDapolyethylenglycol molecule (PEG) at the 5′-end. The internal standardwas brought with buffer to a volume of 360 μl at a concentration of 1μg/μl, and 10 μl each thereof were added to each sample.

To desalt the samples prior to the HPLC analysis NAP-25 columns(Amersham Pharmacia Biotech) were used. The eluates obtained were driedunder vacuum and dissolved in 100 ml of 10 mM Tris-HCl, pH 8.0.

The identification and quantification of the PEGylated spiegelmer wasdone by means of anion exchange chromatography using a Waters Alliance2695 HPLC system and detection at 254 nm. The conditions were asfollows: precolumn: DNAPac PA-100 (504 mm, Dionex) main column: DNAPacPA-100 (2504 mm, Dionex) eluent A: 10 mM NaOH, 1 mM EDTA, 10% (v/v)acetonitril in water eluent B: 375 mM NaCl₄ in eluent A temperature: 25°C. injection volume: 20 μl gradient und flow rates: 0-1 min 10% eluent Bwith 0.5 ml/min; 1-2 min 10% eluent B with 2 ml/min; 2-3 min 30% eluentB with 2 ml/min; 3-13 min 60% eluent B with 2 ml/min; 13-19 min 10%eluent B with 2 ml/min.

The concentration of PEGylated GnRH binding DNA spiegelmer at thedifferent points in time of sampling is shown in FIG. 27. The half timeof the PEGylated GnRH binding DNA spiegelmer upon intravenous injectionis about 4 hours in rats.

EXAMPLE 8 Pharmacokinetics Profile of Unmodified and PEGylated L-RNA inRats

nucleotide sequences:

(SEQ ID NO: 4) L-RNA, 40mer (NOX_M039) 5′ uaa gga aac ucg guc uga ugcggu agc gcu gug cag agc u 3′ (SEQ ID NO: 5) 40 kDalton PEG-L-RNA, 40 mer(NOX_M041) PEG 5′uaa gga aac ucg guc uga ugc ggu agc gcu gug cag agc u3′

The pharmacokinetical profile of the non-PEGylated L-RNA (NOX_M039) andPEGylated L-RNA (NOX_M041) was examined in male rats (CD®, Charles RiverGermany GmbH; weight 280-318 g). After a 7 day settling-in period, 3animals per substance received a single dose of 150 mmol/kg appliedintravenously. 4 rats each per substance received 150 mmol/kg each as asingle subcutaneous dose. The substances were dissolved in 1×PBS pH 7.4(stock solution: 383 μM). After intravenous dose blood samples weretaken for the unmodified L-RNA prior to substance application (0 min)and 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h after substanceapplication and transferred into EDTA Eppendorf tubes for analysis.After intravenous dose blood samples were taken for the PEGylated L-RNAprior to substance application (0 min) and 5 min, 30 min, 1 h, 3 h, 8 h,16 h, 24 h, 36 h as well as 48 h after substance application andtransferred into EDTA Eppendorf tubes for analysis. In subcutaneouslytreated animals blood samples were taken for the unmodified L-RNA priorto substance application (0 min) and 5 min, 15 min, 30 min, 1 h, 2 h, 4h, 6 h after substance application and transferred into EDTA Eppendorftubes for analysis. In subcutaneously treated animals blood samples weretaken for the PEGylated L-RNA prior to substance application (0 min) and5 min, 30 min, 1 h, 3 h, 8 h, 16 h, 24 h, 36 h and 48 h after substanceapplication and transferred into EDTA Eppendorf tubes for analysis.

The amount of L-RNA and PEGylated L-RNA in the blood samples wasexamined by means of a hybridisation assay (see Drolet, D. W. et al.(2000) Pharmacokinetics and safety of an anti-vascular endothelialgrowth factor aptamer (NX1838) following injection into the vitreoushumor of rhesus monkeys. Pharmaceutical Res 17 (12): 1503-1510.). Thehybridisation assay is based on the following principle: the L-RNAmolecule to be detected is hybridised to an immobilised L-DNAoligonucleotide probe (=capture probe; here: 5′-CCG CAT CAG ACC GAG TTTCCT TA T TTT TTT TT-(C7) NH2-3′ (SEQ ID NO: 6)) and detected by abiotinylated detection L-DNA probe (=detector probe; here: 5′-(BB) TTTTTT TT A GCT CTG CAC AGC GCT-3′ (SEQ ID NO: 7)). For this astreptavidine alkaline phosphatase conjugate is bound to the complex ina further step. After addition of a chemiluminescence substrate, lightis generated and measured in a luminometer.

Immobilisation of the oligonucleotide probe: 100 g of the capture probe(0.75 pmol/μl in coupling buffer: 500 mM Na₂HPO₄ pH 8.5, 0.5 mM EDTA)per well were transferred into DNA BIND plates (COSTAR) and incubatedover night at 4° C. Subsequently, it was washed with 3×200 μl couplingbuffer each and incubated for 1 h at 37° C. with 200 μl blocking buffer(0.5% (w/v) BSA in coupling buffer) each. After renewed washing with 200μl coupling buffer and 3×200 μl hybridisation buffer 1 (0.5×SSC pH 7.0,0.5% SDS (w/v)) the plates may be used for detection.

Hybridisation and detection: a 20 pmol/μl solution of the detectionL-DNA probe (=detector probe) in 10 mM Tris-Cl pH 8.0 was prepared. 10μl EDTA plasma (or ddH₂O) were mixed with 90 μl hybridisation buffer 1(0.5×SSC pH 7.0, 0.5% (w/v) SDS). Subsequently, 2 μl of the detectorprobe solution (20 pmol/μl) were added, mixed and centrifuged. Adenaturing step at 95° C. for 10 min in the Thermocycler (MJ Research)followed. The batches were transferred into the DNA-BIND wells preparedaccordingly (see above) and incubated for 2 h at 50° C. Thereafterwashing steps followed: 2×200 μl hybridisation buffer 1 (0.5×SSC pH 7.0,0.5% (w/v) SDS) and 3×200 μl 1×TBS/Tween 20 (20 mM Tris-Cl pH 7.6, 137mM NaCl, 0.1% (v/v) Tween 20). 1 μl streptavidine alkaline phosphataseconjugate (Promega) was diluted with 5 ml 1×TBS/Tween 20. 100 μl of thediluted conjugate were added per well and incubated at room temperaturefor 30 min. Washing steps followed: 1×200 μl 1×TBS/Tween 20 and 3×200 μl1×assay buffer (20 mM Tris-Cl pH 9.8, 1 mM MgCl₂). Finally, 100 μl CSPD“Ready-To-Use Substrate” (Applied Biosystems) were added, incubated 30min at room temperature, and the chemiluminescence was measured in aPOLARstar Galaxy multidetektion plate reader (BMG Labtechnologies).

The concentration-time-curves of the PEGylated L-RNA upon intravenousand subcutaneous dose are shown in FIG. 28 a and FIG. 28 c. Theconcentration profiles of the unmodified L-RNA upon intravenous andsubcutaneous dose are shown in FIG. 28 b and FIG. 28 d. Upon intravenousdose the terminal half time is 50 minutes for the unmodified L-RNA. Forthe PEGylated substance, by contrast, a half time of around 18 hoursresults. Upon subcutaneous dose the terminal half time is 84 minutes forthe unmodified L-RNA, for the PEGylated substance, by contrast, resultsa very long elimination phase.

Thus it is shown, that the modified L-nucleic acid according to theinvention is of advantage in comparison with the unmodified L-nucleicacid. This advantage arises also with a view of the state of the art,described for example by Watson S. R. et al., Antisense nucleic aciddrug dev. 10. 63-75 (2000). In this publication a 2′-F-modified aptameris examined, which binds to L-selectin. The pharmacokinetical half timeof the PEGylated 2′-F-aptamer (40 kDa PEG) administered intravenously invivo in Sprague-Dawley rats is 228 min and is thus clearly shorter thanthose of the L-nucleic acids modified according to the invention.

EXAMPLE 9 General Method for the PEGylation of L-Ribonucleic Acids

A L-ribonucleic acid was generated for the examination of thepharmacological profile of unmodified and PEGylated L-RNA in rats. TheL-RNA has the following sequence:

(SEQ ID NO: 4) 5′-UAA GGA AAC UCG GUC UGA UGC GGU AGC GCU GUG CAG AGCU-3′

The synthesis of the L-RNA with the sequence shown above was performedon an ÄKTA Pilot 10 Synthesizer (Amersham Pharmacia Biotech, Uppsala,Sweden) in a 20 μM scale at a 1000 Å CPG solid phase according to the2-cyanoethyl phosphoramidit chemistry. Subsequently,6-(monomethoxytritylamino)-hexyl-(2-cyanoethyl)-(N,N-diiospropyl)-phosphoramiditwas coupled to the 5′-end of the L-RNA (5′-MMT-aminohexyl-L-RNA) toallow the post-synthesis conjugation with PEG.

After completion of the synthesis the 5′-MMT-aminohexyl-L-RNA wascleaved from the solid phase by 30 min incubation in 41% methylaminesolution at 65° C., and the nucleobases were deprotected completely.Deprotection of the 2′-position was done by incubation in 1.5 ml DMSO,0.75 ml triethylamine (TEA) and 1 ml TEA 3HF for 2 h at 60° C. A firstpurification was done by means of RP-HPLC. The cleavage of themonomethoxytrityl protection group was carried out with 80% acetic acidin 70 min at RT. Acetic acid was removed by two time co-evaporation withethanol, and the 5′-aminohexyl-L-RNA according to SEQ ID NO: 4 purifiedby precipitation in ethanol (yield: 220 OD, 60% pure). The product wastaken up into 1 M sodium acetate, pH 8.0, and desalted by means of sizeexclusion chromatography by a Sephadex G10 column or by Vivaspin 3000(Vivascience, Hannover, Germany).

The L-RNA 5′-amino modified in such a manner (530 OD, 60% pure) wasprepared in aqueous universal buffer according to Theorell and Stenhagen(33 mM sodiumcitrate, 33 mM sodium phosphate, 57 mM sodium borate, pH7.5) (7.5 ml), warmed to 37° C., DMF (5 ml) added, and powderyN-hydroxysuccinimidyl (NHS)-activated ester of branched 40,000 Dapoly(ethylen)glycol was added in portions (2 eq every 45 min,alltogether 18 eq). The progress of the reaction was monitored byanalytical gelectrophoresis (8% polyacrylamide, 8.3 M urea) oranalytical ion exchange HPLC. The raw product was purified initially byion exchange HPLC from excess PEG (Source Q; solvent A: 10 mM sodiumhydrogencarbonate, pH 7.5, solvent B: 10 mM sodium hydrogencarbonate, pH7.5, 2 M sodium chloride, loading of the column and elution of free PEGwith 5% B; flow rate,20 ml/min; separation and elution of the PEG-L-RNAconjugate from non-reacted L-RNA with a gradient up to 35% B over 20column volumes; flow rate 50 ml/min), subsequently desalted byultrafiltration (Labscale TFF System, Millipore). By lyophilisation thedesired product was obtained as a white powder (254 OD, 48% (80% relatedto the purity of the starting product)).

Analogously, further L-nucleic acids including the sequence according toSEQ ID NO:1 were linked with different PEG (linear 10,000 Dalton, linear20,000 Dalton, branched 20,000 Dalton, linear 35,000 Dalton), andpurified.

EXAMPLE 10 Activity Test of a GnRH Binding DNA Spiegelmer in vivo inMale Orchidectomised Rats

Male rats were orchidectomised, whereby the LH level of the ratsincreased steadily during the following eight days due to the missingtestosteron feedback signal. On day 8 the PEG-GnRH DNA spiegelmer (NOX1255) was administered subcutaneously to five rats (100 mg/kg). Bloodsamples were taken on day 0 (prior to the orchidectomy), on day 8 (0hours prior to s.c. application of the GnRH spiegelmer), as well as 0.5h, 1.5 h, 3 h, 6 h 24 h post s.c. application and the respective LHlevel determined using radioimmunoassay (RIA). In parallel, only thevehicle (PBS buffer, pH 7.4) as a negative control was administered s.c.to five male orchidectomised rats, and the standard antagonistCetrorelix (100 μg/kg) s.c. as a positive control to five maleorchidectomised rats. The result is shown in FIG. 29.

The LH levels are lowered in the GnRH DNA spiegelmer group (in FIG. 29depicted as circles) and reach their lowest point after 1.5 h, and stayon for around 3 h. This reduction is comparable to non-orchidectomisedrats and those rats, respectively, treated with Cetrorelix (standardantagonist). Six hours after GnRH DNA spiegelmer dose the LH levelsincrease slowly and reach the level of the untreated control groupwithin 24 h.

Thus the biological effect of the GnRH DNA spiegelmer is observable overa period of 3 hours, while the PEGylated GnRH DNA spiegelmer is activeover a period of 24 hours (see example 5).

The references given in the following correspond to the citations,provided with superscript numbers, given herein.

LITERATURE

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The features of the invention disclosed in the description above, theclaims as well as the figures may be essential individually as well asin any combination for the realisation of the invention in its differentembodiments.

1. A modified L-nucleic acid, comprising an L-nucleic acid part and anon-L-nucleic acid part, wherein the L-nucleic acid part is conjugatedwith the non-L-nucleic acid part, wherein the conjugate of the L-nucleicacid part with the non-L-nucleic acid part has an increased retentiontime in an organism compared to an L-nucleic acid comprising only theL-nucleic acid part, wherein said L-nucleic acid part is a spiegelmer,and wherein said L-nucleic acid part comprises SEQ ID NO:1.
 2. Themodified L-nucleic acid of claim 1, wherein the non-L-nucleic acid parthas a molecular weight of more than about 300 Da.
 3. The modifiedL-nucleic acid of claim 1, wherein the modified L-nucleic acid has amolecular weight to 500,000 Da.
 4. The modified L-nucleic acid of claim1, wherein the L-nucleic acid part has a molecular weight to 50,000 Da.5. The modified L-nucleic acid of claim 1, wherein the non-L-nucleicacid part is Linked to the L-nucleic acid part via a functional group ofthe L-nucleic acid part, wherein the functional group is selected fromthe group consisting of terminal and non-terminal phosphates, terminaland non-terminal sugar portions, natural and non-natural purine bases,and natural and non-natural pyrimidine bases.
 6. The modified L-nucleicacid of claim 5, wherein the linkage of the non L-nucleic acid part withthe L-nucleic acid part is via the 2′-OH—, 3′-OH—, 5′-OH-group or aderivative therefrom, or one or more sugars of the L-nucleic acid part.7. The modified L-nucleic acid of claim 5, wherein the linkage is via atleast one of the positions 5 or 6 of a pyrimidine base.
 8. The modifiedL-nucleic acid of claim 5, wherein the linkage is via a purine base. 9.The modified L-nucleic acid of claim 5, wherein the linkage is at one ormore of the exocyclic amine groups, endocyclic amine groups or ketogroups of a purine or pyrirnidine base or a basic position.
 10. Themodified L-nucleic acid of claim 1, wherein the non-L-nucleic acid partis selected from the group consisting of linear poly (ethylene) glycol,branched poly (ethylene) glycol, hydroxyethyl starch, a peptide, aprotein, a polysaccharide, a sterol, polyoxypropylene, polyoxyamidate,poly (2-hydroxyethyl)-L-glutamine and polyethylene glycol.
 11. Themodified L-nucleic acid of claim 1, wherein a linker is arranged betweenthe L-nucleic acid part and the non-L-nucleic acid part.
 12. Themodified L-nucleic acid of claim 11, wherein said linker is a6-aminohexylphosphate at the 5′-OH end.
 13. The modified L-nucleic acidof claim 12, wherein polyethylene glycol is coupled to the free amine ofthe aminohexyiphosphate linker.
 14. A pharmaceutical compositioncomprising the modified L-nucleic acid of claim 1 and a pharmaceuticallyacceptable carrier, excipient or diluent.
 15. A method for preparing themodified L-nucleic acid of claim 1, comprising the steps: (a) providingan L-nucleic acid comprising SEQ ID NO: 1; (b) providing a non-L-nucleicacid; (c) reacting the L-nucleic acid from (a) and the non-L-nucleicacid from (b); and (d) optionally isolating the modified L-nucleic acidobtained in step (c) wherein the L-nucleic acid part is a Spiegelmer.16. The method of claim 15, wherein the L-nucleic acid in step (a)comprises a linker.
 17. The method of claim 15, wherein after providingthe L-nucleic acid instep (a), a linker is provided.
 18. The modifiedL-nucleic acid of claim 2, wherein the molecular weight is more thanabout 20,000 Da.
 19. The modified L-nucleic acid of claim 18, whereinthe molecular weight is more than 40,000 Da.
 20. The modified L-nucleicacid of claim 8, wherein said linkage occurs at the 8 position.